EndoFree Plasmid Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Plasmid purification method versus transfection efficiency.

Two independent pRSVcat preparations obtained with each method shown were each transfected twice into COS-7, HeLa, and LMH cells using a liposome-mediated method and into Huh7 cells using calcium phosphate. Average transfection efficiencies are expressed as percentages relative to the efficiency obtained with DNA prepared using the QIAGEN Plasmid Kit (100%).

QIAGEN Plasmid Kit procedures.

Neutralized bacterial lysates are incubated directly in the QIAfilter Cartridge and cleared in seconds by filtration. After the endotoxin removal step, the cleared lysate is then loaded onto the anion-exchange tip where plasmid DNA selectively binds under appropriate low-salt and pH conditions. RNA, proteins, metabolites, and other low-molecular-weight impurities are removed by a medium-salt wash, and ultrapure plasmid DNA is eluted in high-salt buffer. The DNA is concentrated and desalted by isopropanol precipitation and collected by centrifugation.

Plasmid purity versus transfection efficiency.

Effect of the amount and quality of plasmid DNA on transfection efficiency in CHO SSF3 X9 cells grown in suspension under serum-free conditions is shown. Each point represents the mean of three independent experiments; Rel. LU: relative light units. (Data kindly provided by M. Zang-Gandor, Novartis AG, Basel, Switzerland.)

For purification of low-copy plasmids and cosmids, the EndoFree Plasmid Mega Kit is a better choice than the EndoFree Giga Plasmid Kit, due to the large culture volumes required and the limited capacity of the QIAfilter Mega-Giga Cartridge.

EndoFree Plasmid Kits remove bacterial endotoxins which are released during the lysis step and influence transfection of DNA into primary cells and sensitive cultured cells. The endotoxin-free DNA obtained from the EndoFree Plasmid Kits is highly suited for reproducible and reliable results in transfection (see figures Plasmid purification method versus transfection efficiency and Plasmid purity versus transfection efficiency and tables "Endotoxin levels in plasmid preparations" and "EndoFree DNA yields high transfection efficiencies with primary cells"). QIAGEN ultrapure endotoxin-free DNA is also suitable for gene therapy research and other sensitive applications.

* Primary rabbit gastric parietal cells were transfected with pEGFP-N2 (CLONTECH) prepared by the methods indicated. Transfections were performed using Effectene Transfection Reagent. The data represent the percentage of cells expressing GFP as determined by scoring the number of green fluorescent cells 48 hours post transfection. The transfection efficiencies represent the average from 6 to 9 replicate dishes from more than 2 different DNA preps for each purification method. (Data kindly provided by C. Chew and J. Parente, Department of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia, USA.)

QIAfilter Cartridges, provided in QIAfilter, HiSpeed, and EndoFree Plasmid Kits, are special filter units designed to replace centrifugation following alkaline lysis of bacterial cells. QIAfilter Cartridges completely remove SDS precipitates and clear bacterial lysates in a fraction of the time needed for centrifugation, reducing plasmid-purification time by up to 1 hour. QIAfilter Mega-Giga Cartridges operate with house vacuum to efficiently clear even large volumes of bacterial lysate with minimal effort (please note that the bottle is not included in the kits). QIAfilter Maxi Cartridges have a syringe-format and lysates are cleared in a matter of seconds by pushing the liquid through the filter.

The unique anion-exchange resin in QIAGEN-tips is developed exclusively for the purification of nucleic acids. Its exceptional separation properties result in DNA purity equivalent or superior to that obtained by two successive rounds of CsCl gradient centrifugation. Prepacked QIAGEN-tips operate by gravity flow and never run dry, minimizing the hands-on time required for plasmid preparation. The entire QIAGEN plasmid purification system avoids the use of toxic substances such as phenol, chloroform, ethidium bromide, and CsCl, minimizing hazard both to the user and the environment.

Endotoxins, also known as lipopolysaccharides or LPS, are cell-membrane components of Gram-negative bacteria such as E. coli (see figure "Bacterial cell wall"). Endotoxins are released during the lysis step of plasmid purification and significantly reduce transfection efficiencies in endotoxin sensitive cell lines (see figures "Plasmid purification method versus transfection efficiency" and "Plasmid purity versus transfection efficiency" and tables "Endotoxin levels in plasmid preparations" and "EndoFree DNA yields high transfection efficiencies with primary cells"). Furthermore, endotoxins can influence the uptake of plasmid DNA in transfection experiments by competing with DNA for “free” transfection reagent. Endotoxins also induce nonspecific activation of immune responses in immune cells such as macrophages and B cells, which can lead to misinterpretation of transfection results. These responses include induced synthesis of proteins and lipids such as IL-1 and prostaglandin. Overall, endotoxins represent a noncontrollable variable in transfection experiment setup, influencing the outcome and reproducibility of results and making them difficult to compare and interpret. In gene therapy research, endotoxins can interfere by causing endotoxic-shock syndrome and activation of the complement cascade.

Specifications

Features

EndoFree Plasmid
Giga Kit

EndoFree Plasmid
Mega Kit

EndoFree Plasmid
Maxi Kit

Applications

Research on gene therapy, transfection of sensitive cells

Research on gene therapy, transfection of sensitive cells

Research on gene therapy, transfection of sensitive cells

Culture volume/starting material

2.5 liters culture volume

500 ml – 2.5 liters culture volume

100–250 ml culture volume

Plasmid type

High-copy, low-copy, cosmid DNA

High-copy, low-copy, cosmid DNA

High-copy, low-copy, cosmid DNA

Processing

Manual (vacuum)

Manual (vacuum and centrifugation)

Manual (vacuum)

Sample per run

1 sample per run

1 sample per run

1 sample per run

Time per run

310 min

220 min

150 min

Yield

<10 mg

<2.5 mg

<500 µg

Procedure

Bacterial cells are lysed under alkaline conditions and the crude lysates are cleared using the QIAfilter Cartridge. At this stage, the Endotoxin Removal Buffer is added to the filtered lysate, which is incubated on ice. The cleared lysate is then loaded onto the anion-exchange tip where plasmid DNA selectively binds under appropriate low-salt and pH conditions. RNA, proteins, metabolites, and other low-molecular-weight impurities are removed by a medium-salt wash, and ultrapure plasmid DNA is eluted in high-salt buffer (see flowchart "QIAGEN Plasmid Kit procedures"). The DNA is concentrated and desalted by isopropanol precipitation and collected by centrifugation.

Applications

DNA purified with EndoFree Plasmid Kits is suitable for any sensitive application, including: